Kaposi's sarcoma herpes virus (KSHV) also known as human herpes virus 8 (HHV-8) causes several cancers including Kaposi's sarcoma, primarily in immune-compromised patients. HHV-8 has been shown to be transmitted sexually, through saliva, and through infected blood and organs to recipients. While a number of serological assays have been devised in the research setting to detect HHV-8 infection, there are no FDA-approved assays. Furthermore, there is no gold standard diagnostic assay for HHV-8 infection and none are of adequate sensitivity and specificity to be used commercially for diagnostic or blood screening purposes. Nevertheless, these assays have revealed that as much as 3 to 5% of US blood donors have been infected with HHV-8, and some high-risk populations such as homosexual men, the HHV-8 prevalence is as high as 65%. Thus, there is a need for a commercial diagnostic to identify HHV-8 infected individuals to prevent further transmission of this virus within the general population and immune-compromised individuals as well as into the general blood supply. The overall goal of this project is to develop a sensitive and specific serological multi-antigen assay for the detection of HHV-8 antibodies that can be used commercially to diagnose at-risk patients and identify blood/tissue donors with HHV-8 infection. Epiphany's first-generation HHV-8 enzyme- linked immunosorbent assay diagnostic (ELISA), composed of a single antigen assay and a dual antigen assay, provide >80% sensitivity and 96% specificity in identifying HHV-8 infected individuals and thus one of the more sensitive diagnostics. However, to be a commercial success, the sensitivity and specificity needs to be improved which we propose by improving the quality and number of capture antigens. In our Phase I grant, we demonstrated the feasibility of our approach by identifying an improved K8.1 capture antigen expressed in a eukaryotic system. To further optimize the assay for commercial use, we will in Aim 1 expand our search for both improved and new capture antigens, then in Aim 2 we will develop a multi-antigen assay. In Aim 3 we will conduct pilot-scale production and beta testing of the diagnostic assay to determine the specificity and sensitivity of the assay by retrospective testing of clinical blood samples. Achieving the goals in this project would generate a commercial HHV-8 diagnostic test which will identify infected patients and donors to reduce transmission risk and to make the blood supply safer.